Abstract: Objective To optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E.coli. Methods The adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column. Results According to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column. Conclusion The optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E.coli.